SARS‐CoV‐2 contains a positive single‐stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS‐CoV and SARS‐CoV‐2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex‐vivo structural probing experiments. These elements contain non‐base‐paired regions that potentially harbor ligand‐binding pockets. Here, we performed an NMR‐based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1H‐based 1D NMR binding assays. The screening identified common as well as RNA‐element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS‐CoV‐2.

RNA cleaving tris(2-aminobenzimidazoles) have been attached to DNA oligonucleotides via disulfide or amide bonds. The resulting conjugates are effective organocatalytic nucleases showing substrate and site selectivity as well as saturation kinetics. The benzimidazole conjugates also degrade enantiomeric RNA. This observation rules out contamination effects as an alternative explanation of RNA degradation. The pH dependency shows that the catalyst is most active in the deprotonated state. Typical half-lifes of RNA substrates are in the range of 12-17 h. Thus, conjugates of tris(2-aminobenzimidazoles) can compete with the majority of metal-dependent artificial nucleases.

2-aminopyridine and 2-aminobenzimidazole were chosen as structural analogues to substitute guanidinium groups in receptor molecules designed as phosphoryl transfer catalysts. Shifting the pKa of the guanidinium analogues toward 7 was expected to raise catalytic activities in aqueous buffer. Although the pKa's of both heterocycles are similar (6.2 and 7.0), only 2-aminobenzimidazole led to active RNA cleavers. All cleavage assays were run with fluorescently labeled substrates and a DNA sequencer. RNase contaminations would degrade RNA enantioselectively. In contrast, achiral catalysts such as 9b and 10b necessarily induce identical cleavage patterns in RNA and its mirror image. This principle allowed us to safely rule out contamination effects in this study. The most active catalysts, tris(2-aminobenzimidazoles) 9b and 10b, were shown by fluorescence correlation spectroscopy (FCS) to aggregate with oligonucleotides. However, at very low concentrations the compounds are still active in the nonaggregated state. Conjugates of 10b with antisense oligonucleotides or RNA binding peptides, therefore, will be promising candidates as site specific artificial ribonucleases.

T ypical features that characterize the catalytic site of natural enzymes are the placement of several functional groups in appropriate relative positions, the control of their solvation, and the recognition of the substrate. They are achieved via protein folding into precise secondary and tertiary conformations. Cofactors such as metal ions may provide additional, and in many instances critical, elements for performing the catalytic process and͞or impart further structural rigidity or conformational control. Cooperativity is the key motif for achieving the astonishing rate accelerations observed by using the functional groups present in protein amino acids that, taken individually, show modest or no reactivity at all (1).Although the design of peptides that fold into well defined tertiary structures mimicking native proteins has been reported recently for sequences of relatively modest size, the exploitation of these structures for the obtainment of efficient catalysts is still in its infancy (2). Rapid progress is being made, however. Notable examples taken from the most recent literature are those provided by Baltzer and coworkers (3, 4), Shogren-Knaak and Imperiali (5), Ghadiri and coworkers (6, 7), and Chmielewski and coworkers (8).We recently reported that peptide sequences as short as 7 aa may adopt an essentially helical conformation in water (9) provided at least five C ␣ -tetrasubstituted ␣-amino acids are present. Furthermore, dinuclear Zn II heptapeptides incorporating two copies of a 1,4,7-triazacyclononane (Tacn)-functionalized amino acid showed clear evidence of cooperativity in the cleavage of RNA model substrate 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP; refs. 10 and 11) and plasmid DNA (12) as well, with rate accelerations in this latter case in the order of 10 million-fold over the uncatalyzed reaction. These peptides set a clear principle: to obtain a cooperative catalyst it is not necessary to design a large molecule. However, they lack a specific recognition site, the number of cooperating functional units is limited, and there is little possibility to incorporate control units such as allosteric regulators (1, 13).For these reasons we decided to move a step forward in the design of a de novo catalyst by increasing the complexity of the system and maintaining to a minimum the synthetic effort required. Because of our interest in synthetic phosphatases (10-12), we focused on a system incorporating catalytic units typically present in this class of enzymes (14-16), i.e., two or more functional metal centers. As an additional feature, we wanted also a regulatory metal ion to provide an allosteric control element (13) in analogy with the role played by Mg II in alkaline phosphatases (17). Materials and MethodsDetails for all of the synthetic procedures, compounds characterization, and analytical techniques used are published as supporting information on the PNAS web site, www.pnas.org. Results and DiscussionDesign. The dissection of the structural features outlined above led us to identify ...